During normal speech, human vocal folds sustain more than 100 high impact collisions each second. Voice overuse may generate nodules on the outer layer of the vocal folds—the so-called superficial lamina propria (“SLP”). In other cases, pathological conditions may render part of the tissue cancerous. In any case, whether the tissue is damaged by overuse or by surgical removal of cancerous tissue, the resulting scar tissue lacks the pliability of the original tissue and voice quality is often seriously reduced.
Over the past few years, significant research effort has been directed toward using tissue engineering approaches to regenerate vocal fold vibratory tissue that responds as normal SLP. As a first step toward developing suitable replacement materials it is important to understand the mechanical response of the natural tissue that the materials will replace, as well as the mechanical response of replacement materials at frequencies of human phonation—approximately 100-900 Hz.
In one known method, the viscoelastic properties of human vocal folds were measured by subjecting the full thickness of the vocal folds (i.e. the lamina propria (LP)) to torsional oscillations in a parallel-plate rheometer. See, for example, R. W. Chan and I. R. Titze, “Viscoelastic Shear Properties of Human Vocal Fold Mucosa: Measurement Methodology and Empirical Results,” J. Acoust. Soc. Am., No. 106, 1999, pp. 2008-2001; and R. W. Chan and I. R. Titze, “Viscoelastic Shear Properties of Human Vocal Fold Mucosa: Theoretical Characterization Based on Constitutive Modeling,” J. Acoust. Soc. Am., No. 106, 2000, pp. 565-580. From these experiments the frequency-dependent storage (shear) modulus and viscosity of the LP over frequencies ranging from 0.01 to 10 Hz was obtained. Notably, this frequency range lies well below the frequency range of human phonation. Measurements at 15 Hz did not follow the trend lines of the results at lower frequencies and were deemed, by the authors, to be “marginally acceptable”.
An important conclusion from the Chan and Titze (1999, 2000) experiments is that the shear modulus of human vocal folds is very low. For most subjects the shear modulus G1 at a frequency of 10 Hz ranges from approximately 10 to 100 Pa. The elastic shear wave speed cs=√{square root over (G1/ρ)} for a material with a shear modulus G1=100 Pa and a density ρ=1000 kg/m3 is approximately 30 cm/s. Because this wave speed is so small relative to wave speeds in most solid materials, one can expect that strong limitations will be imposed on the maximum frequency for which the mechanical properties of the sample can be inferred by means of the usual interpretation of rheometric tests—based on assuming that the stress is nominally uniform through the thickness of the sample. For solid samples the latter assumption holds when the time required for roundtrip transit of stress waves through the thickness of the sample is much less than the period of a single oscillation, i.e. for
                    f        ⪡                              c            s                                2            ⁢            h                                              (        1        )            where h is the sample thickness and f is the driving frequency. For human LP with a thickness h=0.03 cm, as used by Chan and Titze (1999, 2000), the limitation (1) becomes f<<500 Hz or, say, f<30 Hz. Even lower limits on allowable frequencies are obtained for samples with shear moduli near the lower limits of the range of measured values. Moreover, the SLP is known to be more compliant than the intermediate and deep layers of the LP so the measured shear moduli for the full thickness of the LP are expected to be higher than those for the SLP.
Analogously, for fluid samples, a requirement that stresses due to sample inertia are small relative to those due to sample viscosity leads to the limitation (See, for example, H. Schlichting, Boundary Layer Theory, 4th Ed., McGraw-Hill, New York, 1960)
                    f        ⪡                              2            ⁢            η                                2            ⁢            πρ            ⁢                                                  ⁢                          h              2                                                          (        2        )            where η is the viscosity of the sample. From viscosity measurements of Chan and Titze (1999, 2000) the frequency dependent viscosity of human LP can be described approximately by η=η0f−0.85 with η0=1.0 Pa·s. Substitution of this expression for η into (1.2) gives the limitation f<<83 Hz or, say, f<10 Hz.
Whether the frequency limitation is obtained from a constraint of type (1) or (2) it appears that the frequencies for which the viscoelastic properties of human SLP can be measured by standard rheometric methods are likely to be below the frequencies of human phonation. Other methods for measuring viscoelastic properties at high frequencies include electromagnetic torsion methods (see Brodt, et al., “Apparatus for Measuring Viscoelastic Properties Over Ten Decades: Refinements, Review of Scientific Instruments, No. 66, 1995, pp. 5292-5297); electromechanical tensile test methods (R. J. Hemler et al., “A New Method for Measuring Mechanical Properties of Laryngeal Mucosa,” Eur. Arch. Otorhinolaryngol No. 258, 2001, pp. 130-136); and stress-controlled rheometer methods at low audio frequencies (I. R. Titze, et al., “Methodology for Rheological Testing of Engineered Biomaterials at Low Audio Frequencies,” J. Acoust. Soc. Am., No. 115, 2004, pp. 392-401). While all of these methods have attractive features they all neglect wave propagation in the sample. Consequently, they all have frequency limitations similar to those of equations (1) and (2), although the upper limit on allowable frequencies may be extended by using smaller samples.
An alternative approach, based on the analysis of longitudinal waves in viscoelastic cylindrical rods, has been introduced (see Jia, et al., “Synthesis and Charaterization of in situ Crosslinkable Hyaluronic Acid-Based Hydrogels With Potential Applications for Vocal Fold Regeneration,” Macromolecules, No. 37, 2004, pp. 3239-3248) to determine the viscoelastic properties of photo-cross-linked hydrogels. An acoustic shaker was used to subject the base of the rod to an oscillatory vertical motion. The motion of the free, top end of the rod was monitored using a laser-Doppler vibrometer. From the measured amplification factor (the amplitude of the velocity at the top of the rod divided by the amplitude of the velocity at the shaker surface), and the phase shift between the top and bottom ends of the rod, the wave propagation solution was used to determine the frequency dependent viscoelastic moduli for the hydrogel. While this method could extend the measurement of viscoelastic properties into the range of phonation frequencies, it could not be used for LP because the LP geometry does not allow the preparation of slender cylindrical specimens.
Accordingly, those skilled in the art desire methods and apparatus for accurately assessing the suitability of candidate materials for use in human vocal fold reconstruction. Since the accurate assessment of the suitability of candidate materials for use in vocal fold reconstruction requires knowledge of the viscoelastic properties of human vocal fold tissue at human phonation frequencies, those skilled in the art also desire methods and apparatus for accurately assessing the viscoelastic properties of human vocal fold materials at phonation frequencies.
In addition, those skilled in the art desire methods and apparatus for accurately assessing the viscoelastic properties of human vocal fold tissues that can be used with practical tissue samples. In order to be feasible, the methods and apparatus must be suitable for use with available tissue sample geometries and dimensions.
Further, efforts are being made to culture human vocal fold material and possibly other living tissues for use in human vocal fold reconstruction. In order to accurately assess the suitability of cultured materials for use in vocal fold reconstruction, an understanding of the effect of the natural environment on tissue growth must be developed. Accordingly, those skilled in the art also desire methods and apparatus for growing human vocal fold tissues in artificial environments similar to natural environments.